Fibrinogen is a soluble serum protein that serves as the source of fibrin in blood to form clots that are critical to hemostasis, which is the ability of the body to control and maintain adequate blood flow after injury to the vascular system. The extensively studied human fibrinogen is a 340,000 dalton protein, which has a complex oligomeric structure that contains three pairs of related polypeptide chains, designated (Aα)2, (B,β)2, γ2 polypeptide chains. Chemical structural analysis and electron microscopy have demonstrated that the protein has a trinodular structure. In particular, two AαBβγ subunits are oriented in an anti-parallel configuration. The amino terminal portions of the six chains are bundled together in a central “E” domain. Two coiled-coil strands extend outward from either side of the E domain to two terminal nodes, the “D” domains. These coiled-coil regions are 110 amino acids long and composed of all three chains. The D domains contain two high affinity Ca2+ binding sites and are involved with the E domain in fibrin polymerization (see, below). Extensive disulfide bridges covalently cross-link the two subunits and stabilize the globular domains. The carboxy-terminal portions of the Aα chains form flexible extensions beyond the D domains. The D domain contains Factor XIIIa crosslinking sites and is the primary site of plasmic digestion during fibrinolysis. Thus, the individual polypeptide chains of human fibrinogen are extensively linked by disulfide bonds to form an elongated dimeric molecule (for reviews, see, e.g., Hawiger, Semin Hematol, 32:99–109 (1995); Doolittle et al., FASEB J, 10:1464–1470 (1996)).
The biology of fibrinogen and clot (thrombus) formation has been extensively investigated in recent years, and a detailed understanding of the cascade of events leading to clot formation has emerged. There are two major activation pathways (cascades) for coagulation: the intrinsic (or contact coagulation) pathway, which requires Factors XII, IX and VIII; and the extrinsic pathway, which involves tissue factor and Factor VIII. Both pathways converge at the point of activating Factor X, the enzyme responsible for converting prothrombin to thrombin, which then cleaves fibrinogen to form fibrin monomers.
The extrinsic pathway is initiated by tissue factor, a ubiquitous cellular lipoprotein, which forms a calcium-dependent complex with Factor VII. Upon complex formation, Factor VII is activated to Factor VIIa, which converts Factor X to Factor Xa. Factor Xa converts prothrombin to thrombin in conjunction with Factor Va, calcium and phospholipid. Prothrombin conversion also occurs on endothelial surfaces and activated platelets, and requires the assembly of a complex between Factor Xa, Factor Va, and prothrombin. This conversion requires the presence of phospholipid and calcium ions.
The intrinsic or contact coagulation pathway, which is normally initiated by platelets, e.g., in a wound drawing blood. The cascade begins with the formation of a complex among Factor XII, high molecular weight kininogen, and prekallikrein. Upon complex formation, Factor XII is cleaved to Factor XIIa. After the stepwise activation of Factors XI, IX, VIII, X, and V, as in the extrinsic pathway, prothrombin is activated to thrombin. Thrombin, which is a trypsin-like serine protease, is the central regulator of hemostasis and thrombosis.
Fibrin is derived from fibrinogen, and polymerization of fibrin occurs following enzymatic cleavage of fibrinogen by thrombin. Fibrin formation from fibrinogen is a spontaneous self-assembly process resulting from the removal of fibrinopeptides by thrombin. Specifically, thrombin cleavage at the Arg16-Arg17 bond in the Aα chains and at the Arg14-Gly15 bond on the Bβ chains of fibrinogen releases fibrinopeptides A and B, and exposes the fibrin polymerization site in the E domain consisting mainly of the amino (N)-terminus of the α chain. This N-terminus, which bears the amino acid sequence Gly-Pro-Arg-Val (SEQ ID NO:1), binds to a complementary polymerization site on two adjacent fibrinogen chains. End-to-end association of these fibrin molecules mediated by the D domains creates a binding site for the E domain polymerization site, located on a third fibrin molecule. This DD(E) ternary complex forms a core that stabilizes the forming fibrin gel. The initial polymerization product is a linear, two-stranded protofibril. Lateral coalescence of these protofibrils results in thick fibers and a branched, three-dimensional matrix (weak clot). Lateral assembly is complex but probably involves the B polymerization site (the N-terminus of β) and trimolecular complexes formed through D domain interactions.
Adjacent fibrin monomers within the fibrils become covalently cross-linked by Factor XIIIa, a plasma transglutaminase, which is itself activated by thrombin and fibrin and which cross-links lysine 406 and glutamate 398 or glutamate 399 on opposing carboxy-terminal segments of the γ chain (see, e.g., Chen and Doolittle, Biochemistry, 10:4487–4491 (1971); Purves et al., Biochemistry, 26: 4640–4646 (1987). Such crosslinks add mechanical stability to the fibrin network (clot) and increase resistance to clot degradation. Factor XIIIa also enhances clot stability by cross-linking specialized proteins to fibrin, including the plasmin inhibitor α2 antiplasmin and the adhesion protein fibronectin.
The presence of fibrinogen always provides the potential for clot formation as soon as a source of factors of either the intrinsic or extrinsic pathways is provided. Accordingly, methods to isolate, detect, or label this critical component of clot formation would be useful in a variety of fields. Conversely, the potential for clotting is particularly undesirable when blood or plasma must be stored or efficiently passed through an apparatus without clogging the apparatus or without contaminating the blood or plasma with clots, which would present a risk of occlusion leading to an ischemic event, as in stroke or myocardial farct, when the blood or plasma is subsequently returned or later administered to an individual. In such cases, a method to sequester or deplete fibrinogen from a mixture, such as blood or plasma, or from a surface, such as an apparatus or storage container, is imperative to avoid a risk of health and life.
Accordingly, needs remain for the means and methods to efficiently bind fibrinogen for a variety of purposes.